The mitochondrial NADH:ubiquinone oxidoreductase (Complex I) is a key essential component of the bioenergetic machinery in all aerobic cells. The mammalian Complex I is the largest component of mitochondrial respiratory chain;composed of at least 46 individual subunits and up to 10 distinct redox centers. The structural complexity of the enzyme is consistent with its varied catalytic and regulatory properties. Two slowly equilibrating forms of the enzyme, A (active) and D (deactive) exist under given metabolic conditions and each form has different sensitivity to inhibitory and/or activating ligands. This project is to extend our previous collaborative studies on the Complex I A/D transition at the molecular and physiological level. The mechanisms responsible for the inhibitory effects of free fatty acids on Complex I and other physiologically relevant membrane perturbing compounds will be studied. The inhibitory and/or activating effects of fatty acids and their derivatives will be correlated with their modulating effects on the A/D transition and superoxide production by Complex I. These studies will be done in intact mitochondria and inside-out tightly coupled submitochondrial particles. Experiments designed to identify other matrix-located redox enzyme(s) capable of superoxide and hydrogen peroxide production as related to Complex I activity are planned. This research will be primarily done in the Russian laboratory. The results obtained will be used as a guideline for further elaboration at the physiological level of the A/D transition using a model of ischemia/reperfusion in Langendorf-perfused rat hearts, a technique well developed in the U.S. laboratory. The U.S. laboratory will assist the Russian scientists with the Langendorf-perfused heart model and in identifying particular subunits of Complex I that are involved in the regulatory A/D transition. A two-nucleotide binding site(s) model proposed by the Russian group will be tested with the help of the US laboratory by use of a new tight nucleotide-binding site-directed inhibitor recently discovered in the U.S. laboratory. The biomedical significance of this project is to identify the key players involved in regulation of cellular respiration and in development of mitochondria-related pathologies such as occur during oxygen supply deficiency, type 2 diabetes, and neurodegenerative diseases. Defects in Complex I have been shown to contribute to Parkinson's disease and these studies may shed light on how this important metabolic enzyme is regulated.